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A novel doxycycline inducible autoregulatory plasmid which displays ‘on’/‘off’ regulation suited to gene therapy applications

Abstract

The development of transcriptionally controlled systems which function in eukaryotic cells are important for achieving regulated gene expression in gene therapy. In this study we combined the components of the tetracycline-inducible system in self-contained retroviral and plasmid vectors. Regulated reporter gene expression from the autoregulatory plasmid pGTRTL in response to doxycycline (Dox) induction surpasses the expression observed from other self-contained retroviral and plasmid vectors. Induction kinetics and expression levels of luciferase and the therapeutic molecule, truncated soluble complement receptor 1 (sCR1) were characterised in a mouse fibroblast and a human neuroblastoma cell line. The regulatory characteristics of the plasmids were shown to be optimal for gene therapy applications, as there was a rapid reduction in expression levels following removal of Dox. Co-transfection of cells with an autoregulatory plasmid and a Dox inducible enhanced green fluorescent protein (EGFP) plasmid demonstrated the feasibility of using this plasmid combination to achieve parallel regulation of two genes of interest in a single cell under the control of Dox. These novel autoregulatory plasmids display the requirements for gene therapy applications in chronic conditions which are remitting/relapsing such as rheumatoid arthritis or multiple sclerosis, where novel protein therapeutics and combination therapies are needed.

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Acknowledgements

The Wellcome Trust UK, The Arthritis Research Campaign UK, The British Council and Fundacion Antorchas (Argentina) provided financial support for this work. We are grateful to Professor H Bujard for providing the tetracycline plasmids pUHG17–1, pUHD10–3 and pUHC13–3; to Professor D Fearon for providing both mAb YZ1 and control sCR1, and to Dr R Smith for supplying sCR1 rabbit antisera.

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Gould, D., Berenstein, M., Dreja, H. et al. A novel doxycycline inducible autoregulatory plasmid which displays ‘on’/‘off’ regulation suited to gene therapy applications. Gene Ther 7, 2061–2070 (2000). https://doi.org/10.1038/sj.gt.3301354

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